A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be important to gain a detailed understanding of both the nature of the immune response to the current vaccine candidates as well as the immunological status of individuals living in areas in African where malaria is endemic. Over the reporting period this project represented a collaborative effort between Dr. Pierce and Dr. Louis Miller and his colleagues in the Malaria Vaccine Development Unit (MVDU) and with scientists at the Malaria Research and Training Center (MRTC) at the University of Mali. [unreadable] [unreadable] The hallmark of adaptive immunity is antigen-specific immunological memory. Immunological memory is a phenomenon that having been exposed to a pathogen and survived the infection the experience is remembered by the immune system such that upon re-exposure to the same pathogen an individuals immune response is more rapid and stronger such that the individual may experience no clinical systems of the infection. Indeed, all vaccines are predicated on the phenomenon of immunological memory. However, despite its importance we still have an incomplete understanding of the cellular and molecular mechanisms that underlie the generation, maintenance and activation of immunological memory. Clearly, current efforts to develop a malaria vaccine would benefit enormously from an in-depth understanding of the nature of an effective immune response to the parasite that causes malaria. Over the last year we have focused our efforts on gaining an understanding of the generation, maintenance and activation of B cell memory in response to vaccination and to natural malaria infection.[unreadable] [unreadable] In humans, B cell memory is encoded both in long-lived memory B cells and in plasma cells that reside in the bone marrow. The mechanisms by which memory B cells or plasma cells are generated and maintained over a life time are not known. Current evidence, primarily from serological epidemiological studies, indicates that immunological memory to malaria is slow to be acquired, incomplete and short lived. Thus, despite nearly constant exposure to P. falciparum from birth from infectious mosquito bites, children in endemic areas do not acquire immunity that protects them from severe disease until the age of five. Consequently, children under five years of age are susceptible to severe disease that accounts for over 5 million deaths each year in Africa alone. Acquisition of immunity that protects against severe disease but not against mild disease is acquired during adolescence and an immunity sufficient to prevent disease but not to eliminate parasites is acquired only in early adulthood. Our current hypothesis is that P. falciparum infection disrupts the normal mechanisms by which B cell memory is generated, maintained or activated. [unreadable] [unreadable] We have taken advantage of recent advances in the identification of antigen-specific human memory B cells in peripheral blood to describe the generation and maintenance of malaria-specific memory B cells in the U.S. in response to vaccines currently under development in the MVDB and in an endemic area in Mali in response to natural infection. Over the last year, in collaboration with the MVDB, we described the acquisition of antigen-specific memory B cells in the peripheral blood of 15 volunteers enrolled in a trial of the malaria vaccine composed of P. falciparum apical membrane antigen 1 (AMA1) on alum either alone (7 individuals) or in combination with the TLR9 agonist, CpG (8 individuals). Volunteers received three doses of the vaccine 28 days apart and peripheral blood samples were collected 3, 7, 14 and 28 days after each vaccination and up to 200 days following the third and last vaccination. Memory B cells were identified by the method of Ahmed and colleagues that relies on the unique ability of memory B cells, as compared to naive B cells, to respond to a mixture of CpG, pokeweed mitogen and SAC by proliferating and differentiating into clones of antigen-specific antibody secreting cells as measured by ELISPOT assays. We learned that memory B cells are not detected in the peripheral blood until 14 days following the second vaccination and that the frequency of memory B cells peaks by day 28 and is maintained at that level for over 200 days despite a third vaccination. The inclusion of CpG in the AMA1/Alum vaccine resulted in nearly a five fold increase in the number of AMA1-specific memory B cells but the kinetics and persistence of the response were similar. The pattern of the acquisition of memory B cells correlated with the antibody response. These are the first data on the naive human memory B cell response to vaccination and will serve as a baseline for similar analyses in endemic areas. [unreadable] [unreadable] Over the last year we have also initiated studies to determine the kinetics of the acquisition of memory B cells with age and the impact of malaria infection on the acquisition and maintenance of memory B cells. To do so we are conducting a longitudinal study on a cohort of 225 volunteers, 2-25 years of age, in a village outside of Bamako, the capital city of Mali. The study was initiated in June 2006 prior to the malaria transmission season which runs July through December. This transmission season with six months of malaria exposure and six months free of malaria offers a near ideal condition to evaluate the impact of malaria infection on B cell memory generation and maintenance. Peripheral blood samples were collected every two months and 14 days after the first case of malaria for each individual. Our field data shows 298 cases of uncomplicated malaria with a predicted gaussian distribution of malaria cases over the transmission season peaking in October. The proportion of individuals who were malaria free decreased in a clear age dependent fashion during the course of the season. A determination of the frequency of hemoglobin types in a multiple linear regression analysis including age and gender showed a surprising finding that an AS hemoglobin type was associated with significant delay to the first malaria infection. This observation will be important to take into account in vaccine trials that assess time to first malaria case as a measure of immunity. The volunteers G6PD types were also determined and showed no correlation with risk of uncomplicated malaria. Taken together these results indicate that we have acquired quality clinical data on which to interpret the results of our B cell analyses. We are continuing to follow this cohort through the 2007 malaria seasons collecting peripheral blood samples every two months.[unreadable] [unreadable] We plan to assay peripheral blood mononuclear cells collected from the study for the presence of memory B cells by the Crotty-Ahmed assay. Preliminary data on 15 individuals at one time point from the cohort showed that the number of AMA1-specific and tetanus-specific memory B cells correlated with antibody titers. Interestingly, there was no correlation between the number of MSP-1-specific memory B cells and MSP-1 specific antibody titers hinting that there may be a disruption of the maintenance of MSP-1 B cell memory. When completed these studies will provide the first comprehensive analysis of the impact of malaria on the generation and maintenance of memory B cells.